Long noncoding RNA NONHSAT160169.1 promotes resistance via hsa-let-7c-3p/SOX2 axis in gastric cancer

In clinical trials involving patients with HER2 (ERBB2 receptor tyrosine kinase 2) positive gastric cancer, the efficacy of the HER2-targeted drug lapatinib has proven to be disappointingly poor. Under the persistent pressure exerted by targeted drug therapy, a subset of tumor cells exhibit acquired drug resistance through the activation of novel survival signaling cascades, alongside the proliferation of tumor cells that previously harbored mutations conferring resistance to the drug. This study was undertaken with the aim of elucidating in comprehensive detail the intricate mechanisms behind adaptive resistance and identifying novel therapeutic targets that hold promise in the development of effective lapatinib-based therapies for the specific subset of patients afflicted with gastric cancer. We have successfully established a gastric cancer cell line with acquired lapatinib resistance, designated as HGC-27-LR cells. Utilizing comprehensive coding and noncoding transcriptome sequencing analysis, we have identified key factors that regulate lapatinib resistance in HGC-27 cells. We have compellingly validated that among all the lncRNAs identified in HGC-27-LR cells, a novel lncRNA (long noncoding RNA) named NONHSAT160169.1 was found to be most notably upregulated following exposure to lapatinib treatment. The upregulation of NONHSAT160169.1 significantly augmented the migratory, invasive, and stemness capabilities of HGC-27-LR cells. Furthermore, we have delved into the mechanism by which NONHSAT160169.1 regulates lapatinib resistance. The findings have revealed that NONHSAT160169.1, which is induced by the p-STAT3 (signal transducer and activator of transcription 3) nuclear transport pathway, functions as a decoy that competitively interacts with hsa-let-7c-3p and thereby abrogates the inhibitory effect of hsa-let-7c-3p on SOX2 (SRY-box transcription factor 2) expression. Hence, our study has unveiled the NONHSAT160169.1/hsa-let-7c-3p/SOX2 signaling pathway as a novel and pivotal axis for comprehending and surmounting lapatinib resistance in the treatment of HER2-positive gastric cancer.

Gastric cancer exhibits approximately 20% amplification of the ERBB2 receptor tyrosine kinase 2 (HER2) gene and overexpression of the HER2 protein 1 .Multiple novel drugs targeting HER2 drugs are currently being developed for gastric cancer, such as small molecular kinase inhibitors, monoclonal antibodies, antibody-drug conjugates, and other novel therapeutics 2 .One of the classical HER2-targeted kinase inhibitors is lapatinib; it exhibits the advantages of oral administration and favorable patient compliance, and is currently undergoing clinical trials for the treatment of gastric cancer 3 .Lapatinib is a dual inhibitor that competitively binds to the ATP-binding site of the EGFR/HER2 heterodimer, thereby blocking EGFR and HER2 tyrosine kinase activity and inhibiting cell proliferation 4 .
While molecular-targeted therapy holds promise as a therapeutic strategy for enhancing the overall survival (OS) of select patients, drug resistance poses a significant obstacle and remains a limiting factor in treatment outcomes 5 .Currently, patients with HER2-positive gastric cancer who exhibit persistent activation of mitotic signaling pathways are unresponsive to lapatinib treatment; this phenomenon is referred to as primary resistance 6 .However, the possibility of secondary drug resistance in drug-responsive patients remains a concern, as it may result in a decrease or loss of treatment effectiveness within the optimal time frame, which is brief 7 .Under prolonged exposure to targeted drug therapy, some tumor cells may develop adaptive drug resistance by altering their survival signals, in conjunction with the proliferation of cancer cells that originally possessed mutations contributing to drug resistance 8 .
The association between long non-coding RNAs and drug resistance in tumor-targeted therapy is currently a hot topic of research [9][10][11] .LncRNAs are aberrantly expressed at different stages of gastric cancer progression, and a few studies have reported mechanisms by which lncRNAs regulate lapatinib-induced drug resistance in gastric cancer; hence, lncRNAs associated with drug resistance may potentially be targeted for personalized therapeutic strategies [11][12][13] .In this study, through transcriptome microarray, we aimed to identify novel lncRNAs associated with lapatinib resistance as well as key genes that interact with lncRNAs in gastric cancer to establish a signal regulatory network.Our findings may aid in predicting drug sensitivity in patients with gastric cancer and devising tailored treatment strategies for individual patients.

Scratch wound healing assay
HGC-27 or HGC-27-LR cells were seeded in six-well plates and cultured until they were 100% confluent.Scratch wounds were created by dragging a 200 μL pipette tip into cell monolayers.Before replacing the conditioned medium, cells were washed three times with 1 × PBS (0.1 M PBS, pH 7.4) to eliminate non-adherent cellular debris.Cell migration to the wound surface was monitored and subsequently measured for distance.

Transwell assay
In brief, 2 × 10 5 cells were seeded into the upper chambers of a transwell (TCS-013-024, 8.0 μm; BIOFIL) and suspended in serum-free medium overnight.The upper compartment of transwell filter inserts contained serumfree culture medium, while the lower compartment contained culture medium supplemented with 15% serum with or without lapatinib.Following incubation at 37 °C and 5% CO 2 , cells were fixed with paraformaldehyde for 30 min, washed three times with 1 × PBS, stained with crystal violet (0.1%) for 30 min, washed with 1 × PBS to remove the excess dye, dried at room temperature, and then photographed.

Quantitative real-time PCR (qPCR)
Total RNA was extracted using Trizol (9109; TAKARA).RNA was reverse-transcribed to cDNA using the Pri-meScript RT Reagent Kit (DRR047; TAKARA).The reaction volume for qPCR was 20 μL comprising 10 μL of SYBR qPCR Master Mix (Q321-02; Vazyme), forward primers, reverse primers, template cDNA, and nucleasefree H 2 O.The relative mRNA level of a specific gene was normalized to that of beta-actin.The primer sequences are provided in the supplementary material (Table S1).

Construction of stable knockdown or overexpression cells
Lentivirus packaging of vector, NONHSAT160169.1, shRNA-NC, shRNA-NONHSAT160169.1, and SOX2 were supplied by GeneChem Co., Ltd.(Shanghai, China).HGC-27 or HGC-27-LR cells were seeded in six-well plates (1 × 10 5 cells per well) and cultured in high-glucose DMEM (D5796; Sigma) supplemented with 10% FBS.The plasmids and the infection reagent (HiTransG P) were thoroughly mixed and added to the wells.Following incubation at 37 °C with 5% CO 2 for 16 h, the medium was replaced with fresh medium.After 48-72 h, the transfection efficiency was determined.Then, stable cell lines were established by screening with puromycin (2 μg/mL).

In vivo xenograft assay
Statement: All animal experiments were performed in accordance with the requirements of the American Veterinary Medical Association (AVMA) Animal Euthanasia Guidelines.NCG mice (female, 5 weeks; Gempharmatech Co., Ltd.) were randomly divided into four groups (n = 8 per group).The density of LV-vector-HGC-27, LV-NONHSAT160169.1-HGC-27,shRNA-NC-HGC-27-LR, or shRNA-NONHSAT160169.1-HGC-27-LR cells were adjusted to 7 × 10 6 cells/100 μL PBS and then subcutaneously injected into each NCG mouse following anesthetization by isoflurane inhalation.At the end of this experiment, mice were placed in euthanasia cages, which were then filled with CO 2 at a balanced rate to render them unconscious quickly and minimize their suffering as much as possible.During euthanasia, the breathing condition and eye color of each mouse were continuously observed.The mice were not taken out of the cage until they stopped breathing and lost their eye color.Tumor tissues were isolated and collected from each mouse.Xenograft tumors were analyzed to determine their volume and weight and then photographed.Tumor volume was calculated as follows: tumor volume = length × width 2 × 0.52.All animal experiments were performed in accordance with the ARRIVE guidelines and approved by the Animal Ethics Experiment Committee of Xuzhou Medical University (202112A435).

Immunohistochemistry (IHC)
Xenograft tumors were fixed in 4% paraformaldehyde, and then paraffin-embedded tissue sections were prepared.The tissue sections were deparaffinized and then boiled in citrate buffer (0.01 mol/L, pH 6.0) for 20 min to conduct antigen retrieval.Tissues were permeabilized with 0.3% Triton X-100 for 20 min at room temperature (not required for membrane proteins).The sections were then washed three times with 1 × PBST for 5 min, blocked with 10% goat serum for 1 h at room temperature, and then incubated with primary antibodies against Ki-67 (1:200; 12202S; Cell Signaling Technology) or CD31 (1:1200; 3528S; Cell Signaling Technology) overnight at 4 °C.The sections were then washed three times with 1 × PBST for 5 min, and the activity of endogenous HRP was blocked using 0.3% H 2 O 2 .The sections were then washed three times with 1 × PBST for 5 min, followed a incubation with secondary antibody for 1 h at 37 °C.The sections were washed three times with 1 × PBST for 5 min and reacted with diaminobenzidine (DAB) for 1-10 min.The sections were then washed with hematoxylin for 3 min and rinsed with distilled water.Next, the sections were subjected to dehydration, and the slices were sealed.Finally, sections were observed and photographed using a multispectral high-throughput IHC scanning system (Olympus, Japan).

Statistical analysis
Experimental data are presented as means ± standard error of mean for at least three independent experiments.All statistical analyses were performed using SPSS 17.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA).P < 0.05 was considered to indicate statistical significance.

Ethics statement
All the animal experiments were performed in accordance with the ARRIVE guidelines and approved by the Animal Ethics Experiment Committee of Xuzhou Medical University (202112A435).At the end of the experiment, we euthanized the mice in strict accordance with the requirements of the American Veterinary Medical Association (AVMA) Animal Euthanasia Guidelines.

Migration, invasion, and stem cell phenotypes were enhanced in lapatinib-resistant HER2-positive gastric cancer cells
The HGC-27 cell line has been widely used in studies on gastric cancer progression, improvements in therapeutic strategies, and drug resistance [14][15][16] .HGC-27-LR cells were established using a classical approach to subjecting cells to long-term drug gradient pressure in vitro 9 .Subsequently, CCK-8 analysis was performed to determine any potential alterations in the sensitivity of HGC-27 cells to lapatinib.The results suggested that lapatinib inhibited the growth of HGC-27 cells in a concentration-dependent manner, while no such effect was observed in HGC-27-LR cells.The half-maximal inhibitory concentrations (IC 50 ) of lapatinib for the inhibition of HGC-27 and HGC-27-LR were 5.807 μM and 32.755 μM, respectively.The resistance index (RI) was 5.64 (RI = IC 50 -HGC-27-LR cell/IC 50 -HGC-27 cell) (Fig. 1a).Compared to parental cells, the migration and invasive abilities of HGC-27-LR cells were significantly enhanced (Fig. 1b-e).Drug resistance due to alterations in the cell stemness phenotype during cancer treatment has been widely reported [17][18][19] .The sphere formation assay revealed that individual HGC-27-LR cells exhibited a greater capacity for self-renewal and cellular aggregation into clusters compared to individual HGC-27 cells (Fig. 1f).In addition, the levels of CD133 (prominin 1) and ALDH1A1 (aldehyde dehydrogenase 1 family member A1) proteins, which are markers for gastric tumor stem cells, were notably increased in HGC-27-LR cells (Fig. 1g).Altogether, these results indicated the successful establishment of a lapatinib-resistant cell line, which exhibited enhanced migration, invasion, and stemness phenotypes.

Lapatinib-induced pressure triggered lncRNA NONHSAT160169.1 upregulation, which promoted migration, invasion, self-renewal abilities, and lapatinib resistance of HGC-27 cells
To identify novel noncoding RNAs and survival signaling mechanisms that contribute to lapatinib resistance in HGC-27 cells, we performed transcriptome microarray analysis for both coding and noncoding RNAs.Compared to HGC-27 cells, lncRNA NONHSAT160169.1 in HGC-27-LR cells was most significantly increased in the microarray, with a fold change of 714.8 (Fig. 2a,b).NONHSAT160169.1 was identified as a lncRNA using the NONCODE database (http:// www.nonco de.org/ index.php).This lncRNA is located in the forward strand of chromosome 11 (hg38) and has a total length of 1384 bp (Table S3).However, despite a search of the NCBI database, no relevant information or research pertaining to NONHSAT160169.1 was found.Therefore, this novel lncRNA needs to be investigated further.Initially, we analyzed the level of NONHSAT160169.1 in both HGC-27 and HGC27-LR cells and its level under lapatinib treatment.The results indicated that NONHSAT160169.1 was highly expressed in HGC-27-LR cells (Fig. 2c) (only the data of NONHSAT160169.1, which was closely associated with this study are shown; validation results of other candidate target genes are not shown).RNA-FISH analysis revealed that NONHSAT160169.1 was distributed in both the nucleus and cytoplasm of HGC-27 and HGC-27-LR cells.Moreover, the fluorescence intensity of NONHSAT160169.1 was markedly increased in HGC-27-LR cells (Fig. 2d).

NONHSAT160169.1 induced stemness of HGC-27 cells by upregulating SOX2 expression
The aforementioned results indicated that an individual HGC-27-LR cell possessed a greater capacity for selfrenewal and proliferation into cell clusters (Fig. 1f).Since NONHSAT160169.1 was a key factor that promoted lapatinib resistance in HGC-27 cells, we investigated whether NONHSAT160169.1 could affect the stem-like phenotype of cells that stably overexpress NONHSAT160169.1 or cells in which NONHSAT160169.1 was knocked down.The results showed that the overexpression of NONHSAT160169.1 enhanced the stemness of HGC-27 cells, whereas the knockdown of NONHSAT160169.1 reduced the stemness of HGC-27-LR cells (Fig. 4a).To understand the mechanisms by which NONHSAT160169.1 promotes HGC-27-LR cell stemness, we performed transcriptome microarray sequencing of all coding genes in both HGC-27 and HGC-27-LR cells.Among the top 20 notably upregulated coding genes in HGC-27-LR cells, SOX2 attracted our attention, which was positively correlated with NONHSAT160169.1 (Fig. 4b).Studies have shown that SOX2 promotes tumor cell stemness and plays a crucial role in triggering resistance to cancer therapies [20][21][22][23] .We hypothesized that NONHSAT160169.1 increased lapatinib resistance in HGC-27 cells by inducing SOX2 expression.qPCR and western blot analysis revealed that mRNA and protein levels of SOX2 were increased in HGC-27 cells following NONHSAT160169.1 overexpression.Similarly, the expression of SOX2 was decreased in HGC-27-LR cells in which NONHSAT160169.1 was knocked down (Fig. 4c,d).The expression of SOX2 was then compared between www.nature.com/scientificreports/HGC-27 and HGC-27-LR cells.The results showed that the expression of SOX2 was increased in HGC-27-LR cells (Fig. 4e,f).Additionally, using the Online Kaplan Meier Plotter database (https:// kmplot.com/ analy sis/), we found that high expression of SOX2 was positively correlated with short OS in gastric cancer patients.Investigating the effect of SOX2 on lapatinib-resistant HGC-27 cells may hold some clinical significance (Fig. 4g).Hence, these results indicated that NONHSAT160169.1 induced stemness in HGC-27 cells by upregulating SOX2 expression.
Next, we determined the effect of hsa-let-7c-3p on the expression of SOX2.The mRNA and protein levels of SOX2 decreased following transfection of hsa-let-7c-3p mimics into HGC-27-LR cells exhibiting high SOX2 expression (Fig. 5e,g); however, mRNA and protein levels of SOX2 increased following transfection of hsa-let-7c-3p inhibitor into HGC-27 cells (Fig. 5f,h).Therefore, these results validated that hsa-let-7c-3p was negatively modulated and NONHSAT160169.1 promoted the expression of SOX2.

Lapatinib-induced p-STAT3 upregulation promoted the transcription of NONHSAT160169.1 and lapatinib tolerance in HGC-27 cells
We further explored the mechanism underlying NONHSAT160169.1 upregulation in HGC-27-LR cells.Through bioinformatics analysis (http:// jaspar.binf.ku.dk/), we predicted that the STAT3 could bind to the promoter of NONHSAT160169.1.Potential sites in the NONHSAT160169.1 promoter where STAT3 can bind are shown in Fig. 6a.Previous studies have validated that STAT3 is a key downstream signal molecule of EGFR/HER2 and is involved in the regulation of chemoresistance 25,26 .p-STAT3 translocates into the nucleus after phosphorylation and then functions as a transcription factor to mediate the expression of its target genes 27 .Thus, we compared STAT3 and p-STAT3 levels in HGC-27 and HGC-27-LR cells.Western blot analysis revealed that p-STAT3 was markedly increased in HGC-27-LR cells (Fig. 6b).This phenomenon was induced by lapatinib treatment, as the results suggested that lapatinib significantly upregulated the level of p-STAT3 in HGC-27 cells but not HGC-27-LR cells (Fig. 6c).Moreover, analysis of the proteins in the nuclear and cytoplasmic fractions suggested that, compared to that in HGC-27 cells, the nuclear distribution of p-STAT3 in the HGC-27-LR cells was increased (Fig. 6d).Therefore, the increase in NONHSAT160169.1 expression induced by lapatinib was dependent on nuclear translocation of phospho-STAT3 (Tyr705).
We designed 10 pairs of various primers according to the predicted binding sites.ChIP and qPCR analysis validated that could bind most strongly to a − 869 ~ − 653 bp fragment of NONHSAT160169.1 promoter (Fig. 6e).Moreover, agarose gel electrophoresis of − 869 ~ − 653 bp qPCR products showed discernable and distinct bright bands (Fig. 6f).Predicted STAT3 binding site motifs were highly consistent with the predicted sites on the NONHSAT160169.1 promoter (Fig. 6g).Furthermore, to comprehensively understand whether STAT3 was the upstream signaling molecule of NONHSAT160169.1-inducedSOX2 upregulation, we treated HGC-27-LR cells with S3I-201, which is an inhibitor of STAT3 DNA binding activity.Subsequently, the total RNA from HGC-27-LR cells was extracted, and expression levels of NONHSAT160169.1 and SOX2 were determined.The results showed that S3I-201 treatment dramatically decreased the level of NONHSAT160169.1 (Fig. 6h), and the mRNA and protein levels of downstream SOX2 were also significantly downregulated (Fig. S2a,b).
Furthermore, we conducted a comprehensive investigation on the intricate interplay between STAT3 and SOX2 expression, a downstream molecule of NONHSAT160169.1, and the role of STAT3 in regulating the sensitivity of HGC-27 cells to lapatinib by constructing stable overexpression (LV-vector-HGC-27 and LV-STAT3-HGC-27) and knock-down STAT3 (sh-NC-HGC-27-LR and sh-STAT3-HGC-27-LR) cell lines.The overexpression and knockdown efficiency of STAT3 were detected by western blot.The results suggested that the expression of SOX2 increased in LV-STAT3-HGC-27 cells, while in the sh-STAT3-HGC-27-LR cells, the expression of SOX2 reduced (Fig. S3).We next performed CCK-8 analysis to clarify the effect of STAT3 on lapatinib resistance in HGC-27 cells.The results showed that lapatinib sensitivity of HGC-27-LR cells was increased after STAT3 knockdown (Fig. S4a,b).In addition, after overexpression of STAT3, the viability of HGC-27 cell was higher than that of empty vector transfection group under the treatment of different concentrations of lapatinib for 72 h (Fig. S4c,d).The above results suggested that STAT3 aggravated lapatinib tolerance in HGC-27 cells.
Therefore, p-STAT3 was the upstream inducer of NONHSAT160169.1.Long-term lapatinib treatment increased the level of p-STAT3 in HGC-27 cells, accompanied by its nuclear translocation, allowing it to bind to the NONHSAT160169.1 promoter and induce its expression.Moreover, the expression of SOX2 downstream of NONHSAT160169.1 was increased.In conclusion, we elucidated a novel mechanism underlying lapatinib resistance in gastric cancer from the perspectives of lncRNA, miRNA, and mRNA using genome-wide gene chip technology for high-throughput screening.Long-term lapatinib stimulation upregulates the expression of NONHSAT160169.1 via p-STAT3 (Tyr705) in HGC-27-LR cells.As a ceRNA, NONHSAT160169.1 decoys hsa-let-7c-3p and increases the level of SOX2, thereby aggravating lapatinib resistance in HGC-27 gastric cancer cells (Fig. 9).Above all, our study elucidates a novel survival signaling pathway during the progression of adaptive lapatinib resistance in HGC-27 cells, and targeting NONHSAT160169.1 is expected to be a potential therapeutic strategy for overcoming lapatinib resistance in HER2-positive gastric cancer.

Figure 1 .
Figure 1.Migration, invasion, and stemness properties were enhanced in HGC-27-LR cells.(a) HGC-27 and HGC-27-LR cells were treated with various concentrations of lapatinib for 72 h.Cell viability was analyzed by CCK-8 assay, and then the IC 50 value and drug resistance index of cells were calculated.(b) After making a scratch, we treated HGC-27 and HGC-27-LR cells with 5 μM lapatinib for 24 h.The wounds' width was measured, and relative wound closure was quantified.(c) Transwell assays were performed to analyze migration and invasion of HGC-27 and HGC-27-LR cells, respectively.After 24 h, the cells were fixed and stained with crystal violet.Photographs were then captured and the cell counts were determined (scale bar = 100 μm).(d,e) HGC-27 and HGC-27-LR cells were treated with 10 μM lapatinib for 24 h or 48 h.Transwell assays were performed to analyze the migratory and invasive properties of the cells (Scale bar = 100 μm).(f) Sphere formation of HGC-27 and HGC-27-LR cells was analyzed after cultured in six-well ultra-low adhesion culture plates for 7 days.The number of spheres (diameter > 50 μm) was determined.(g) The protein levels of CD133 and ALDH1A1 in HGC-27 and HGC-27-LR cells were determined by western blot.Results are shown as mean ± SEM (*p < 0.05, **p < 0.01).

Figure 6 .
Figure 6.Lapatinib-induced increase in the level of p-STAT3 promoted NONHSAT160169.1 transcription.(a) Using the JASPAR database, we predicted multiple binding sites of STAT3 in NONHSAT160169.1 promoter.(b) Western blot analysis of p-STAT3 and STAT3 in HGC-27 and HGC-27-LR cells.The gray values of the bands were analyzed.(c) HGC-27 and HGC-27-LR cells were respectively treated with 5 μM lapatinib for 24 h.Western blot analysis of the protein levels of p-STAT3 and STAT3.The gray values of the bands were analyzed.(d) Western blot analysis of p-STAT3 in the subcellular fractions of HGC-27 and HGC-27-LR cells (GAPDH: cytoplasmic protein marker; histone H3: nucleus protein marker).(e) ChIP analysis of the direct binding of p-STAT3 to NONHSAT160169.1 promoter detected by using qPCR.(f) Agarose gel electrophoresis of the qPCR product of the most significant DNA fragment obtained from (e) (− 869 ~ − 653 bp).(g) The predicted binding motifs of STAT3 corresponding to the NONHSAT160169.1 promoter are shown.The sequence of predicted binding sites in − 869 ~ − 653 bp were + TTG TTT GAAAA and + ATT TTA TGAAA.(h) In HGC-27-LR cells, the RNA level of NONHSAT160169.1 was detected using qPCR following treatment with S3I-201 for 48 h and 72 h.Results are shown as mean ± SEM (*p < 0.05, **p < 0.01).

Figure 9 .
Figure 9. Schematic of the mechanism by which NONHSAT160169.1 promoted resistance in gastric cancer cells.(a, b) On the basis of the noncoding and coding transcriptome sequencing analyses, we identified key factors involved in the regulation of lapatinib resistance in HGC-27 cells.Long-term treatment with lapatinib promoted p-STAT3 nuclear translocation, thereby upregulating the expression of NONHSAT160169.1, which then enhanced the migration, invasion, and stemness phenotypes of HGC-27 cells.NONHSAT160169.1 decoyed hsa-let-7c-3p as a ceRNA and contributed to reversing the inhibitory effect of hsa-let-7c-3p on SOX2 expression.Therefore, NONHSAT160169.1/hsa-let-7c-3p/SOX2 was shown to be a novel signaling pathway positively regulating lapatinib resistance in HGC-27 cells.